实验海洋生物学重点实验室知识库

牙鲆（Paralichthys olivaceus）是一种广泛养殖的经济鱼类，其面临的病害问题严重限制了养殖业的发展。在本文中，我们针对牙鲆补体系统开展了研究，揭示了两个重要的补体因子，补体因子3（Complement component 3, C3）和补体因子I（Complement Factor I, CFI）在补体激活及抗感染免疫中的作用。

在哺乳动物中，补体系统是主要的先天性免疫机制之一，在宿主防御病原过程中起着重要的作用。其中，C3是补体系统的中心因子，其活化状态影响到整个补体系统的功能发挥。在硬骨鱼类中，C3的研究主要集中在序列鉴定和表达水平上，对于其生物学功能尚不完全清楚。本研究中，我们检测了牙鲆C3 （PoC3）的免疫学功能。我们发现PoC3拥有C3家族成员典型的结构域和高度保守序列。敲降牙鲆PoC3后，补体活性显著降低。血清中的PoC3具有结合细菌的能力，天然纯化的PoC3具有杀伤细菌和结合牙鲆FG细胞的能力。这些结果表明硬骨鱼C3能够参与补体系统的激活，并且在宿主抗感染免疫中发挥重要作用。

在补体调控系统中，CFI是血清中的一种丝氨酸蛋白酶，在补体调控功能中发挥重要作用。在辅因子的参与下，可以将C3b降解为iC3b，进一步降解为C3c和 C3d，以此调控补体的激活，避免补体的过度激活损伤宿主。在本研究中，我们研究了牙鲆CFI （PoCFI）的生物学功能。发现PoCFI由597个氨基酸组成，并且包含一个丝氨酸蛋白酶（PoCFI-Tryp）结构域。PoCFI在牙鲆9个不同的组织中都有表达，并且在细菌的刺激下表达上调。重组表达蛋白PoCFI-Tryp（rPoCFI-Tryp）可以通过降解血清中C3b，抑制补体激活。rPoCFI-Tryp表现出了广谱的细菌结合能力，并能抑制细菌的生长。这些结果首次证明了CFI通过降解C3b负调控补体激活，并可能在宿主免疫防御细菌感染过程中发挥作用。综上所述，通过对牙鲆补体系统的两个重要因子（PoC3和PoCFI）的研究，加深了对硬骨鱼类补体系统抗感染免疫机制的了解，为海水养殖鱼类的免疫防治奠定了基础。

Japanese flounder (Paralichthys olivaceus) is a widely cultivated economic fish. Microbial disease is a severe problem of Japanese flounder aquaculture. In this study, in order to understand the complement system against pathogens, we examined the functions of two important complement factors in complement activation and against pathogens, i.e., complement component 3 (C3) and complement factor I (CFI).

In mammals, complement system is one of the main innate immune mechanisms and plays an important role in host defense against pathogens. C3 is the central factor of complement system, and the activation state of C3 affects the functions of the whole complement system. In teleost, C3 has been cloned and analyzed in expression level, however, the biological function of C3 is poorly understood. In this study, we examined the immunological property of C3 from Japanese flounder (PoC3). We found that PoC3 has typical domains and highly conserved sequences of C3 superfamily. Knockdown of PoC3 in Japanese flounder resulted in significant inhibition of complement activities. In serum, PoC3 interacted directly with bacteria. Purified PoC3 killed bacteria and bound to FG cells of Japanese flounder. These results suggest that the teleost C3 involved in the complement activation and played an important role in host innate immune against infection.

In complement regulation system, CFI is a serine protease and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. CFI limits further complement activation and prevents excessive tissue damage. In this study, we studied the biological function of CFI (PoCFI) of Japanese flounder. PoCFI consists of 597 amino acids and possesses a serine protease (PoCFI-Tryp) domain. PoCFI was expressed in 9 different tissues of Japanese flounder, and its expression was up-regulated by bacterial challenge. The recombinant protein PoCFI-Tryp (rPoCFI-Tryp) can inhibit complement activation by degrading C3b and may play a role in host immune defense against bacterial infection. rPoCFI-Tryp showed a broad spectrum of bacterial binding ability and inhibited bacterial growth. These results demonstrate for the first time that CFI negatively regulates complement activation by degrading C3b and may play a role in host immune defense against bacterial infection. In conclusion, the study of two important complement factors (PoC3 and PoCFI) of Japanese flounder deepened the understanding of the against infection mechanism of teleost complement system, and provided a guidance for the prevention and control of cultured fish.

第1章 绪论.... 1

1.1 补体系统... 1

1.1.1 补体系统的组成... 1

1.1.2 补体系统的激活通路... 2

1.1.3 补体负调控机制... 3

1.2 补体因子C3. 4

1.3 补体调控I因子... 6

1.4 鱼类免疫系统... 7

1.5 鱼类补体研究进展... 8

1.6 本论文研究的目的与意义... 9

第2章 牙鲆重要补体因子C3的功能研究.... 11

2.1 实验材料与方法... 11

2.1.1 实验所用菌株、细胞及动物... 11

2.1.2 实验试剂与实验仪器... 12

2.1.3 序列分析... 13

2.1.4 RNA干扰（RNAi）敲降PoC3. 14

2.1.5 pPoC3si质粒干扰效率的检测... 19

2.1.6 检测干扰PoC3的血清溶血活性... 21

2.1.7 牙鲆PoC3蛋白的天然纯化... 21

2.1.8 PoC3多克隆抗体的制备... 22

2.1.9 ELISA方法检测多克隆抗体的效价... 22

2.1.10 Western blot方法检测抗体的特异性... 22

2.1. 11 Western blot检测血清中PoC3对多种细菌的结合... 23

2.1.12 PoC3的杀菌作用检测... 23

2.1.13 免疫荧光检测C3对FG细胞的结合作用... 23

2.2 实验结果... 24

2.2.1 序列特征分析... 24

2.2.2 PoC3参与牙鲆血清的补体激活... 27

2.2.3 PoC3的天然纯化和Western blot分析... 28

2.2.4血清PoC3结合多种细菌... 29

2.2.5 PoC3的杀菌效应分析... 30

2.2.6 PoC3结合FG细胞... 31

2.3 讨论... 32

第3章 补体负调控因子Factor I的功能研究.... 35

3.1 实验材料与方法... 35

3.1.1 实验菌株与实验动物... 35

3.1.2 实验试剂与实验仪器... 35

3.1.3 序列分析... 35

3.1.4 实时荧光定量PCR检测PoCFI的组织分布... 36

3.1.5 pETPoCFI-Tryp表达载体的构建... 37

3.1.6 表达菌株的构建... 39

3.1.7 rPoCFI-Tryp的表达、纯化和复性... 39

3.1.8 PoCFI-Tryp多克隆抗体的制备... 41

3.1.9 PoCFI-Tryp多克隆抗体效价及特异性检测... 41

3.1.10 rPoCFI-Tryp的溶血活性检测... 41

3.1.11 rPoCFI-Tryp的杀菌活性检测... 42

3.1.12 抗体封闭后检测溶血活性和杀菌活性... 42

3.1.13 Western blot 检测rPoCFI-Tryp对PoC3的降解作用... 42

3.1.14 ELISA检测牙鲆血清中C3b含量... 42

3.1.15 rPoCFI-Tryp与细菌的相互作用... 43

3.1.16 rPoCFI-Tryp抑制细菌生长... 44

3.2 实验结果... 44

3.2.1 序列特征分析... 44

3.2.2 PoCFI的基因表达分析... 46

3.2.3 rPoCFI-Tryp的表达纯化和Western blot分析... 48

3.2.4 rPoCFI-Tryp对补体激活的负调控作用... 49

3.2.5 rPoCFI-Tryp对血清中PoC3b的降解作用... 51

3.2.6 rPoCFI-Tryp结合多种细菌... 51

3.2.7 rPoCFI-Tryp的抗菌效用... 53

3.3 讨论... 54

第4章 结论与展望.... 57

参考文献.... 61

致 谢.... 67

作者简历及攻读学位期间发表的学术论文与研究成果    69